Objective: The aim is to investigate the protective effect of andrographolide (AP) on retinal ganglion cells (RGC) in rats with glaucoma and its mechanism. Methods: ninety-six adult male Wistar rats were randomly divided into normal control (NC) group, chronic ocular hypertension (CHOT) group, vehicle group, and AP group. The density of RGCs and the number of apoptotic cells in retinal slices were evaluated, and the function of RGC was evaluated by photopic negative wave response (PhNR). The expression of Bcl-2, Bax, and caspase-3 in the retina was detected. Results: (1) The density of RGC in the CHOT and vehicle groups were significantly lower than that in the NC and AP (P < 0.01). After AP intervention, the RGC density of the rats significantly increased (P < 0.01). The ganglion cell layer (GCL) of the CHOT and vehicle groups was obviously thinner, and the thickness of the GCL was partially restored in the AP group. (2) A large number of terminal deoxytransferase mediated dUTP nick end labeling (TUNEL) positive cells were found in the GCL of the CHOT and vehicle groups, but only a few TUNEL positive cells were found in the AP group. The percentage of TUNEL positive cells was 8.80 +/- 4.97%, 37.00 +/- 5.27%, 46.16 +/- 6.50%, and 22.29 +/- 3.52% for groups NC, CHOT, vehicle, and AP, respectively. (3) At 2 weeks, the amplitude of PhNR in the CHOT and vehicle groups decreased by 63.22 +/- 13.89% and 57.88 +/- 6.95%, respectively. The amplitude of PhNR in the AP group was only 22.56 +/- 6.44% lower than that in the NC group. The AP intervention significantly reduced the decrease of the PhNR amplitude in CHOT eyes. (4) Compared with the NC group, the expression of anti-apoptotic protein Bcl-2 was decreased in the CHOT and vehicle groups, and the expression of the pro-apoptotic protein Bax and caspase-3 significantly increased in the CHOT and vehicle groups, which could be reversed by AP treatment. The trend of the reverse transcription-polymerase chain reaction was consistent with that of the western blot analysis. Conclusion: The protective effect of caspase-3/Bcl-2 may be achieved through the regulation of the Caspase-3/Bcl-2 pathway in the rat retina.